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Tall fescue seed extraction and partial purification of ergot alkaloids.

Ji H, Fannin F, Klotz J, Bush L - Front Chem (2014)

Bottom Line: Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried.The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline.The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

View Article: PubMed Central - PubMed

Affiliation: Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.

ABSTRACT
Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

No MeSH data available.


Related in: MedlinePlus

Contractile response of bovine lateral saphenous vein. Response of bovine lateral saphenous vein to ergovaline, an endophyte-infected tall fescue seed extract (E+EXT) standardized to ergovaline concentration, and a mixture of ergot alkaloids (ALK) that reflects the alkaloid profile of E+EXT. Regression lines represent the fitting of data to a sigmoidal concentration response curve. The ALK treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this ALK treatment were based on alkaloids quantified in the E+EXT. E+EXT was prepared as described above for purification of the crude extract from the tub extractor. All treatment solutions were based on the ergovaline concentration. The other alkaloids in the E+EXT and the ALK mimic solution were in the 10−8 to 10−10 M. Figure taken from Foote et al. (2012).
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Figure 5: Contractile response of bovine lateral saphenous vein. Response of bovine lateral saphenous vein to ergovaline, an endophyte-infected tall fescue seed extract (E+EXT) standardized to ergovaline concentration, and a mixture of ergot alkaloids (ALK) that reflects the alkaloid profile of E+EXT. Regression lines represent the fitting of data to a sigmoidal concentration response curve. The ALK treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this ALK treatment were based on alkaloids quantified in the E+EXT. E+EXT was prepared as described above for purification of the crude extract from the tub extractor. All treatment solutions were based on the ergovaline concentration. The other alkaloids in the E+EXT and the ALK mimic solution were in the 10−8 to 10−10 M. Figure taken from Foote et al. (2012).

Mentions: Purified extracts were used in experiments to measure the biological activity in bovine vasculature. An experiment was conducted to determine if substances extracted from endophyte infected tall fescue other than ergovaline were responsible for vasculature responses in vitro (Foote et al., 2012). Lateral saphenous veins from cattle were tested in a multi-myograph with different alkaloid treatments. Alkaloid treatments were (1) chemically pure ergovaline (EV), (2) endophyte infected seed extract (E+EXT); (3) a mixture of alkaloids (ALK) that mimicked those found in the E+EXT, and (4) an extract from endophyte free seed extract. An extract was generated using endophyte containing seed described above and a second similar extract was generated from endophyte-free seed. The partially purified residue from each extract was solublized in 50 mL of 80% methanol. The endophyte-free extract was diluted in the same manner as for the extract from the endophyte-infected seed. The amount of each extract and chemical alkaloid treatment added to myograph cells were determined by amount of ergovaline required meet the ergovaline concentration along the X-axis (Figure 5). Ergovaline/ergovalinine ratio of the alkaloid extract was 60/40. A mixed alkaloid treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this mixed alkaloid treatment were based on alkaloids quantified in the endophyte infected seed extract, including ergovaline. Both the E+EXT and ALK mixture induced similar contractile response as ergovaline. Lack of a significant response from the endophyte-free seed extract (data not shown) and the fact that the response from the E+EXT and ALK were similar to the chemically pure ergovaline response (Figure 5) suggests that ergovaline is the only substance in the endophyte-infected tall fescue extract that is causing the contractile response in this bioassay, and perhaps in any of the affected vasculature. The seed extract also decreased in vivo reticuloruminal epithelial blood flow about 50% and volatile fatty acid absorption (acetate, propionate and butyrate) from the washed reticulorumen (Foote et al., 2013). Acute exposure of in vitro bovine rumen epithelium to the extract had no effect on acetate or butyrate flux (μmol/cm2 h) across the epithelium (Foote et al., 2014).


Tall fescue seed extraction and partial purification of ergot alkaloids.

Ji H, Fannin F, Klotz J, Bush L - Front Chem (2014)

Contractile response of bovine lateral saphenous vein. Response of bovine lateral saphenous vein to ergovaline, an endophyte-infected tall fescue seed extract (E+EXT) standardized to ergovaline concentration, and a mixture of ergot alkaloids (ALK) that reflects the alkaloid profile of E+EXT. Regression lines represent the fitting of data to a sigmoidal concentration response curve. The ALK treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this ALK treatment were based on alkaloids quantified in the E+EXT. E+EXT was prepared as described above for purification of the crude extract from the tub extractor. All treatment solutions were based on the ergovaline concentration. The other alkaloids in the E+EXT and the ALK mimic solution were in the 10−8 to 10−10 M. Figure taken from Foote et al. (2012).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263182&req=5

Figure 5: Contractile response of bovine lateral saphenous vein. Response of bovine lateral saphenous vein to ergovaline, an endophyte-infected tall fescue seed extract (E+EXT) standardized to ergovaline concentration, and a mixture of ergot alkaloids (ALK) that reflects the alkaloid profile of E+EXT. Regression lines represent the fitting of data to a sigmoidal concentration response curve. The ALK treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this ALK treatment were based on alkaloids quantified in the E+EXT. E+EXT was prepared as described above for purification of the crude extract from the tub extractor. All treatment solutions were based on the ergovaline concentration. The other alkaloids in the E+EXT and the ALK mimic solution were in the 10−8 to 10−10 M. Figure taken from Foote et al. (2012).
Mentions: Purified extracts were used in experiments to measure the biological activity in bovine vasculature. An experiment was conducted to determine if substances extracted from endophyte infected tall fescue other than ergovaline were responsible for vasculature responses in vitro (Foote et al., 2012). Lateral saphenous veins from cattle were tested in a multi-myograph with different alkaloid treatments. Alkaloid treatments were (1) chemically pure ergovaline (EV), (2) endophyte infected seed extract (E+EXT); (3) a mixture of alkaloids (ALK) that mimicked those found in the E+EXT, and (4) an extract from endophyte free seed extract. An extract was generated using endophyte containing seed described above and a second similar extract was generated from endophyte-free seed. The partially purified residue from each extract was solublized in 50 mL of 80% methanol. The endophyte-free extract was diluted in the same manner as for the extract from the endophyte-infected seed. The amount of each extract and chemical alkaloid treatment added to myograph cells were determined by amount of ergovaline required meet the ergovaline concentration along the X-axis (Figure 5). Ergovaline/ergovalinine ratio of the alkaloid extract was 60/40. A mixed alkaloid treatment was prepared by dissolving ergovaline, ergotamine, α-ergocryptine, ergocristine D-lysergic acid hydrate, and ergonovine in 80% methanol. The alkaloid concentrations of this mixed alkaloid treatment were based on alkaloids quantified in the endophyte infected seed extract, including ergovaline. Both the E+EXT and ALK mixture induced similar contractile response as ergovaline. Lack of a significant response from the endophyte-free seed extract (data not shown) and the fact that the response from the E+EXT and ALK were similar to the chemically pure ergovaline response (Figure 5) suggests that ergovaline is the only substance in the endophyte-infected tall fescue extract that is causing the contractile response in this bioassay, and perhaps in any of the affected vasculature. The seed extract also decreased in vivo reticuloruminal epithelial blood flow about 50% and volatile fatty acid absorption (acetate, propionate and butyrate) from the washed reticulorumen (Foote et al., 2013). Acute exposure of in vitro bovine rumen epithelium to the extract had no effect on acetate or butyrate flux (μmol/cm2 h) across the epithelium (Foote et al., 2014).

Bottom Line: Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried.The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline.The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

View Article: PubMed Central - PubMed

Affiliation: Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.

ABSTRACT
Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

No MeSH data available.


Related in: MedlinePlus